Axons are directed along very stereotyped trajectories towards their targets by the coordinate action of several different mechanisms. Although many axon guidance molecules have been identified, precisely how these proteins function to ensure reproducible and high fidelity guidance in vivo is not clear. The semaphorins comprise one family of axon guidance molecules. The class 3 semaphorins function predominantly as repellents and are differentially expressed in selective motor neuron (MN) subsets in the developing embryo. This raises the possibility that class 3 semaphorins could function as repellents to affect motor axon guidance. The goal of this proposal is to determine the role of class 3 semaphorins in defining motor axon trajectories in vivo. This work will further our understanding of nerve regeneration and our efforts to promote regrowth and repair after nerve injury, as the same mechanisms that control axon growth during development are likely to be important during regeneration. The specific aims are: Aim 1: To characterize the normal distribution of class 3 semaphorins and their receptors in the mouse embryo, semaphorin expression will be localized by in situ hybridization for mRNA and sema-sensitive axons will be identified with alkaline-phosphatase tagged recombinant semaphorins. Aim 2: To map MN pools with regard to class 3 semaphorin responsiveness and test whether a particular semaphorin functions as a repellent for a subset of MNs, we will create a transgenic mouse in which ectopic expression of a class 3 semaphorin is driven by the myoD promoter/enhancer in all muscles. Aim 3: To determine if axonal trajectories are aberrant in semaphorin-deficient mice, axon trajectories of homozygous sema3A, sema3C or sema3A/3C double mutants will be compared with their respective heterozygous and wildtype littermates. The fidelity of axon pathfinding in Aims 2 and 3 will be determined by retrograde labeling and immunohistochemistry.